Differences in diacylglycerol acyltransferases expression patterns and regulation cause distinct hepatic triglyceride deposition in fish

Triglyceride (TAG) deposition in the liver is associated with metabolic disorders. In lower vertebrate, the propensity to accumulate hepatic TAG varies widely among fish species. Diacylglycerol acyltransferases (DGAT1 and DGAT2) are major enzymes for TAG synthesis. Here we show that large yellow croaker (Larimichthys crocea) has significantly higher hepatic TAG level than that in rainbow trout (Oncorhynchus mykiss) fed with same diet. Hepatic expression of DGATs genes in croaker is markedly higher compared with trout under physiological condition. Meanwhile, DGAT1 and DGAT2 in both croaker and trout are required for TAG synthesis and lipid droplet formation in vitro. Furthermore, oleic acid treatment increases DGAT1 expression in croaker hepatocytes rather than in trout and has no significant difference in DGAT2 expression in two fish species. Finally, effects of various transcription factors on croaker and trout DGAT1 promoter are studied. We find that DGAT1 is a target gene of the transcription factor CREBH in croaker rather than in trout. Overall, hepatic expression and transcriptional regulation of DGATs display significant species differences between croaker and trout with distinct hepatic triglyceride deposition, which bring new perspectives on the use of fish models for studying hepatic TAG deposition.

remove the bioinformatic analyses described in this section and instead focus on the novel findings that advance knowledge of gene regulation.3. It is interesting how the expression of hepatic DGAT2 is greater than DGAT1 in both species (Fig 3), yet the protein appears to contribute minimally to oleic acid-and acetic acid-induced TAG formation or lipid droplet formation when overexpressed in cells (Fig 4).Supporting this observation is the OA induced expression DGAT1 but not DGAT2, and inhibition of DGAT2 does not significantly attenuate the OA response (Fig 5).The authors do not address these counter-intuitive findings in the discussion, but it is worthwhile to do so.Can they speculate on the functional significance of such a highly expressed gene (DGAT2) when the functional assays support the lower expressed DGAT1 is more functionally active as a regulator of TAG synthesis?The sentence in lines 261-264 that states DGAT2 is perhaps more significant for species-specific differences should also be reconsidered, given that DGAT1 appears to be more functionally significant than DGAT2.4. Absent throughout the results is a densitometric analysis of protein abundance for the Western blot assays.It is critical for protein abundance to be quantified and statistically analyzed for differential expression.Currently, evaluation of relative protein abundance can is only a subjective evaluation of how dark/thick the bands are.Given that a considerable number of inferences are made from these data, the findings generated from Western blot analyses must be supported by the appropriate analyses.Similarly, a quantitative analysis of lipid droplet number and/size is essential for images in Response: Thanks for the advice.We agreed with the suggestion and revised our title as "Differences in diacylglycerol acyltransferases expression patterns and regulation cause distinct hepatic triglyceride deposition in fish".
3. The bioinformatics section in lines 116-136 does not contribute significantly to the overall findings.Genes have also been annotated and sequences are available in public databases.It is recommended to remove the bioinformatic analyses described in this section and instead focus on the novel findings that advance knowledge of gene regulation.

Response:
We thank the reviewer for this comment and agree.According to the reviewer's suggestion, we simplified the bioinformatic section and deleted some unnecessary sequence analysis.And phylogenetic and motif analysis of DGATs were included in the revised manuscript because it showed that that amino acid sequence of DGATs was generally conserved in the evolution and their function should be conserved.It is consistent with our hypothesis that the function of DGATs in fish are conserved and the gene regulation are different.

It is interesting how the expression of hepatic DGAT2 is greater than DGAT1 in both species (Fig 3), yet the protein appears to contribute minimally to oleic acid-and acetic acid-induced TAG formation or lipid droplet formation when overexpressed in cells (Fig 4)
. Supporting this observation is the OA induced expression DGAT1 but not DGAT2, and inhibition of DGAT2 does not significantly attenuate the OA response (Fig 5).The authors do not address these counter-intuitive findings in the discussion, but it is worthwhile to do so.Can they speculate on the functional significance of such a highly expressed gene (DGAT2) when the functional assays support the lower expressed DGAT1 is more functionally active as a regulator of TAG synthesis?The sentence in lines 261-264 that states DGAT2 is perhaps more significant for species-specific differences should also be reconsidered, given that DGAT1 appears to be more functionally significant than DGAT2.
Response: Thanks for the suggestion.We also surprised with the results we got.However, it is consistent with the results about DGATs function in mammals.As we know, DGAT1 and DGAT2, are the main enzymes responsible for TAG synthesis.And they play specific roles in different tissues and in some species in TAG synthesis, especially on substrate specificity.There is plenty of evidence in mammal researches that DGAT1 and DGAT2 mediate distinct hepatic functions.DGAT2 is primarily responsible for incorporating endogenously synthesized Fatty acids (FAs) into TG, whereas DGAT1 plays a greater role in esterifying exogenous FAs to glycerol.In addition, teleost fish utilize glucose poorly and the capacity of the pathway to convert glucose into cellular lipids for storage is relatively low (5,6).Fish meal and fish oil are the most popular energy resource for the fish.It makes sense that DGAT1 and DGAT2 are active and DGAT1 has stronger effect in fish liver which utilize lots of exogenous FAs.It is consistent that DGAT1 might be very important for hepatic TG synthesis in fish.DGAT2 also has high expression in fish liver, but de novo lipogenesis activity in liver is lower.In our previous publication, we also found DGAT1 inhibition can totally blocked triglyceride accumulation induced by oleic acid in croaker hepatocytes (7).In the end, we improved our discussion in the revised manuscript to make it better.Response: We thank the reviewer for bringing up this important point.Following the suggestion from the reviewer, we provided relative protein abundance analyses in Fig 5 and Fig 8.At the same time, we simplified the results and corrected the legends in the revised manuscript.As for lipid droplet, we tested the lipid droplet formation in specific yeast H1246 cells.The H1246 cells is a triglyceride synthesis-deficit mutant yeast and have no lipid droplet.The goal for using this line is to check whether croaker or trout DGATs is contribute to lipid droplet formation.The contribution of DGATs on lipid droplet formation could be our next scientific question.And there is the limit for quantifying the lipid droplet in yeast.We will check this problem if we get the DGATs-KO cell lines.7.There should be some statistical support provided to support the statement in line 203 about the dose dependent increase in promoter activity.A simple linear regression with suffice.
Response: Thanks for the kind suggestion.Based on the results in fig7B, it implied that the croaker DGAT1 promoter activity increased with the increasing of CREBH concentration.It would support strongly that CREBH upregulate DGAT1 promoter activity in croaker.We improved the sentence in the revised manuscript .

Acetate acid should be written as acetic acid.
Response: Thanks for the suggestion.The word has been revised in the revised manuscript.
Fig 4e. 5. Why are t-tests used in some graphs while multiple means comparisons are used other times?For example, Fig 4a uses a multiple means comparison while Figs 4b-d, despite showing the same type of analysis/data, use a t-test to compare to the control.Please be consistent.In cases where comparing between all treatment groups is valuable (such as Fig 4 or Fig 6a,b), then a multiple means comparison will be most insightful regarding relative contribution of each protein to total activity.6.There should be some statistical support provided to support the statement in line 203 about the dose dependent increase in promoter activity.A simple linear regression with suffice.Minor comments: 1. Acetate acid should be written as acetic acid.2. No where in the manuscript is it indicated what inhibitors of DGAT1 and DGAT2 were used.3. Please improve clarification in the figure legends regarding what two treatments are being compared for t-tests (ie: what treatment is used for the control).In some figures like Fig 6a,b, it is not always obvious. 4. Fig 7: Does CREBH-F mean the full-length transcript, compared to CREBH-N which is the N-terminus of the protein?Some panels in Fig 7 have CREBH while others have CREBH-F.Are they the same thing?Please clarify.
5) Viegas I, Jarak I, Rito J, et al.Effects of dietary carbohydrate on hepatic de novo lipogenesis in European seabass (Dicentrarchus labrax L.) [J].Journal of lipid research, 2016, 57(7): 1264-1272.6) Bou M, Todorčević M, Torgersen J, et al.De novo lipogenesis in Atlantic salmon adipocytes[J].Biochimica et Biophysica Acta (BBA)-General Subjects, 2016, 1860(1): 86-96.7) Xiang X, Han S, Xu D, et al.Effects of DGAT1 inhibition on hepatic lipid deposition, antioxidant capacity and inflammatory response in Larimichthys crocea[J].Aquaculture, 2021, 543: 736967.5. Absent throughout the results is a densitometric analysis of protein abundance for the Western blot assays.It is critical for protein abundance to be quantified and statistically analyzed for differential expression.Currently, evaluation of relative protein abundance can is only a subjective evaluation of how dark/thick the bands are.Given that a considerable number of inferences are made from these data, the findings generated from Western blot analyses must be supported by the appropriate analyses.Similarly, a quantitative analysis of lipid droplet number and/size is essential for images in Fig 4e.

6.
Why are t-tests used in some graphs while multiple means comparisons are used other times?For example, Fig 4a uses a multiple means comparison while Figs 4b-d, despite showing the same type of analysis/data, use a t-test to compare to the control.Please be consistent.In cases where comparing between all treatment groups is valuable (such as Fig 4 or Fig 6a,b), then a multiple means comparison will be most insightful regarding relative contribution of each protein to total activity.Response: We appreciate the reviewer's comment.Sorry for the author' carelessness.We agreed with reviewer 's advice and revised the Fig4 using multiple means comparison.In Fig 4a-d, we want to show relative contribution of each protein to total activity which can help us to know the function of croaker and trout DGATs.And we use the t-test for Fig 6a,b which can show the effect on DGAT1 promoter by each transcriptional factor.

9.
No where in the manuscript is it indicated what inhibitors of DGAT1 and DGAT2 were used.Response: Thank you for your kind suggestion.Sorry for the author' carelessness.According to the reviewer's advice, we supplemented the information of DGAT1 and DGAT2 inhibitor in the revised manuscript (Line 387-388).10.Please improve clarification in the figure legends regarding what two treatments are being compared for t-tests (ie: what treatment is used for the control).In some figures like Fig 6a,b, it is not always obvious.Response: Thanks for the kind suggestion.We improved the figure legends in revised manuscript.In Fig 6a,b, The negative control was the cells transfected with an empty plasmid with no promoter sequence (pGL6-basic) and empty PCS2 plasmid.The value was used to normalize (n=4).11.Fig 7: Does CREBH-F mean the full-length transcript, compared to CREBH-N which is the N-terminus of the protein?Some panels in Fig 7 have CREBH while others have CREBH-F.Are they the same thing?Please clarify.Response: Thanks for the suggestion.Yes.CREBH-F is the full-length transcript.The N-terminal fragment of CREBH (CREBH-N) (amino acids 1-291) was amplified from the full length of CREBH (CREBH-F).We clarified the abbreviation and marked it clear in the Fig 7 in the revised manuscript.